Reducing conditions are the key for efficient production of active ribonuclease inhibitor in Escherichia coli

Abstract

BackgroundThe eukaryotic RNase ribonuclease/angiogenin inhibitors (RI) are a protein group distinguished by a unique structure - they are composed of hydrophobic leucine-rich repeat motifs (LRR) and contain a high amount of reduced cysteine residues. The members of this group are difficult to produce in E. coli and other recombinant hosts due to their high aggregation tendency.ResultsIn this work dithiothreitol (DTT) was successfully applied for improving the yield of correctly folded ribonuclease/angiogenin inhibitor in E. coli K12 periplasmic and cytoplasmic compartments. The feasibility of the in vivo folding concepts for cytoplasmic and periplasmic production were demonstrated at batch and fed-batch cultivation modes in shake flasks and at the bioreactor scale.Firstly, the best secretion conditions of RI in the periplasmic space were evaluated by using a high throughput multifactorial screening approach of a vector library, directly with the Enbase fed-batch production mode in 96-well plates. Secondly, the effect of the redox environment was evaluated in isogenic dsbA+ and dsbA- strains at the various cultivation conditions with reducing agents in the cultivation medium. Despite the fusion to the signal peptide, highest activities were found in the cytoplasmic fraction. Thus by removing the signal peptide the positive effect of the reducing agent DTT was clearly proven also for the cytoplasmic compartment. Finally, optimal periplasmic and cytoplasmic RI fed-batch production processes involving externally added DTT were developed in shake flasks and scaled up to the bioreactor scale.ConclusionsDTT highly improved both, periplasmic and cytoplasmic accumulation and activity of RI at low synthesis rate, i.e. in constructs harbouring weak recombinant synthesis rate stipulating genetic elements together with cultivation at low temperature. In a stirred bioreactor environment RI folding was strongly improved by repeated pulse addition of DTT at low aeration conditions.

Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression

“…These data are in good agreement with our previous data of RI production in the E. coli K-12 strain, were the dependency of the RI activity on the DTT concentration was demonstrated [15]. …”

“…Previously we had improved cytoplasmic and periplasmic folding by either screening for a functioning fusion partner [13] or by controlling the redox situation by external addition of DTT [15] in the E. coli RV308 K-12 strain.…”

Expression and in vitro functional analyses of recombinant Gam1 protein

“…2C and 3). Reducing environment could have decreased the formation of false intermolecular disulfide bonds that can cause dimerization or aggregation [29,30]. Finally, during the multiple trials to optimize the expression and purification of pCold-TF constructs, it was discovered that cooling to 16 °C for a prolonged time (1 h) before induction, increased the production of monomeric Gam1.…”

Increased Bacterial Cell Density and Recombinant Protein Yield Using a Commercial Microbial Cultivation System

“…Strategies range from simple media enrichment or modification (some examples include addition of reducing agents [7,8] or oxygen carriers [9] ) to the use of nutrient-releasing substrates that replicate aspects of fed-batch systems. EnBase (BioSilta) is a commercially available microbial cultivation system that increases cellular yield by utilizing enzymatically controlled release of glucose, from a polymeric substrate, to growth media.…”

“…Previous studies of EnBase media performance have evaluated biomass yield, [11][12][13] protein yield, [2,13] protein solubility, [11,12] capacity for changes in production scale and culture vessel, [3,12,14] media composition and metabolites, [7,9,10] compatibility with high-throughput production, [18] and protein activity. [8] This study assessed the effect of EnBase media compared with Luria-Bertani (LB) media on attributes of bacterial cell yield and protein yield, solubility, and immunoreactivity of two recombinant capripoxvirus proteins.…”